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2017 Cardiovascular Research Day Abstract Book

13 An Unbiased

13 An Unbiased Classification Algorithm for Transverse Tubule Remodeling Within Murine Heart Failure Models. Dylan Colli 1 • Bradley Stewart 2 • Peter Kekenes-Huskey, PhD 2 1Chemical and Materials Engineering, University of Kentucky • 2 Chemistry, University of Kentucky Undergraduate Transverse tubules (TTs) are the main method of delivery of extracellular calcium into the myocyte, responsible for the initiation of calcium induced calcium release (CICR) necessary to carry out excitation contraction coupling (EC coupling) within ventricular myocytes. Disruptions in the regularly striated TT network are correlated with various degrees of calcium mishandling and are generally observed within heart failure models. Currently, TT remodeling is often judged qualitatively, subject to bias of the experimenter. To address the need to eliminate this bias, we propose a technique that utilizes: 1. Computer vision libraries. 2. Signal processing techniques such as matched filtering that provide signal-to-noise ratios, a quantitative measure of observed remodeling. Combining these methods, an unbiased classification algorithm is presented that is able to quantify both regions of TT loss and longitudinal remodeling as well as characterize whole myocytes by the magnitude of their deviation from the prototypical healthy myocyte. This allows for the expedient, and more importantly, unbiased and reliable classification of TT remodeling observed within murine ventricular myocytes. 29

14 Cardiac specific Rad knockout Increases Calcium Release Mihir Shah 1 • Andrea Sebastian 1 • Landon Simpson 2 • Doug Andres, PhD 2 • Jonathan Satin, PhD 1 • Brooke Ahern 1 1Physiology, University of Kentucky • 2 Biochemistry, University of Kentucky Undergraduate Background: The L-type calcium channel complex (LTCC) associates with regulatory proteins including Rad that govern calcium channel function. Overexpression of Rad inhibits LTCC inward calcium current. In contrast, whole body constitutive Rad knockout in mice have higher calcium influx in cardiac myocytes. It is unknown whether the effect of constitutive whole body Rad knockout arise from development, from the influence of non-cardiac myocytes, or is a primary result of Rad reduction in cardiac myocytes. Our recently engineered inducible, cardiac myocyte restricted Rad-knockout mouse model allows us to address these possibilities. Hypothesis: Cardiac specific Rad knockout will recapitulate the same effects of global Rad knockout. Acute induction (1-4 weeks) of Rad knock out produces an immediate effect on cytosolic calcium entry. Methods: Cytosolic Ca2+ in single, live cells were analyzed using Fura-2. Cells were paced at 1 Hz. Twitch calcium, calcium reuptake, and release kinetics were measured before and after the addition of isoproterenol in both wild-type (Radfl/fl abbreviated as WT) and induced, Rad-/- cardiomyocytes (cRadKO). Results: Twitch calcium levels were significantly higher in cRadKO compared to WT (1.99±0.09 and 1.54±0.13 FU for WT and RadKO, respectively, p=0.008). cRadKO and WT twitch calcium significantly increased in response to acute isoproterenol (ISO; 2.63±0.13 and was 1.98±0.20 FU for WT and RadKO, respectively, p=0.0082). Calcium reuptake also increased with the knockout of Rad and in the presence of ISO. The onset velocity of calcium release increased in cRadKO (38.13±3.46 FU/ms and 48.71±2.38 FU/ms, for WT and cRadKO, respectively, p=0.01). The ISO response for onset velocity was retained with cRadKO, in parallel with amplitude effects. Conclusion: Reduction of Rad protein elevated baseline cytosolic calcium levels approaching that of ISO-stimulated WT cardiomyocytes. Thus, Rad knockout provides positive inotropic support to heart function by elevating baseline cytosolic Ca2+. cRadKO maintains modulated myocardial function to sympathetic activity. 30

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